Project Summary RNA modifications embody a crucial layer of epigenetic regulation of gene expression1,2. N6-methyladenosine RNA (m6A) is the most prevalent modification present on eukaryotic mRNA, and has been shown to modulate several post-transcriptional processes such as mRNA splicing, mRNA decay, secondary structure, and translation1,2. Recent studies have uncovered critical roles of m6A modification in developmental processes, cell differentiation, cancer metastasis, and cellular stress responses3-13. Mechanistic studies in mammalian cell lines suggest that m6A deposition is critical during the cellular stress response, where methylation has been shown to control selective translation and stress granule localization8-13,46. However, the role of m6A in cellular stress in vivo and in the aged brain has not been addressed. Given the crucial role of the stress response in brain aging and neurodegenerative disease, m6A modification of RNA may play an important role. To elucidate this role, in Aim 1 I propose to address the functional consequence of increased m6A mRNA modification (seen by dot blot analysis) in the chronic stress of aging in Drosophila brains. This would be the first in vivo analysis of m6A dynamics in the brain during aging. I will assess which specific transcripts show changed levels of m6A modification by performing m6A-miCLIP sequencing from brains of 3-d young and 30-d aged flies. To further understand the consequence of altered modification levels on the transcript I will also examine transcript levels by RNA-seq, and translation efficiency by ribosomal sequencing in control and m6A methyltransferase (Ime4) knockdown fly brains in young and aged flies. Furthermore, my preliminary data suggest that Ime4 knockdown flies cause a decrease in lifespan. I will examine also if Ime4 RNAi flies exhibit increased vacuole degeneration from paraffin sectioning of brains. Aim 2 will examine the role of m6A RNA reader protein CG6422 in neurodegeneration, specifically TDP-43 induced degeneration. Preliminary data shows that TDP-43 expressing fly heads have increased levels of m6A methylated mRNA than controls. Furthermore, ubiquitous reduction of the m6A cytoplasmic reader protein CG6422 in flies which express TDP-43 dramatically reduces lifespan in comparison to TDP-43 expressing flies. It is likely that CG6422 is playing a role in the pathological stress response caused by TDP-43 toxicity, and upregulation of this gene may have therapeutic benefits. To further understand this role, I will examine if the decrease in lifespan also exists with a neuronal specific expression of TDP-43 and CG6422 RNAi, examine retinal degeneration with CG6422 RNAi, and possible interaction of CG6422 with TDP-43. I will further examine the effect CG6422 may be playing on stress regulation processes induced during neurodegeneration such as heat shock protein levels and stress granule dynamics. By examining m6A regulation in aged flies or fly models of degenerative disease, this study will create a better understanding of the functional role of m6A in physiological and pathological stress regulation.